The ultimate goal of this proposal is to determine whether filovirus VP40 regulates the Hippo signaling pathway and tight junction integrity, thereby attenuating host innate antiviral defenses and facilitating vascular leakage/hemorrhaging during filovirus pathogenesis. Exploratory approaches to better understand viral-host interactions that contribute to the transmission and pathogenesis of these deadly viruses are needed more urgently than ever, as Ebola virus can cross the blood-brain barrier and re-emerge months later in the CNS, semen, and other immunologically privileged sites that are inaccessible to antibody therapy. Our laboratory focuses on the mechanisms by which the PPxY L-domain motifs of filovirus VP40 protein interact with and recruit host proteins to regulate late stages of virion production and dissemination. Our exciting preliminary data raise the intriguing possibility that VP40 also mimics and competes with host proteins to regulate both the host innate immune response, and the manifestation of vascular permeability/hemorrhaging and/or invasion of immune privileged sites by filoviruses. We utilized a highly selective WW-domain protein array to identify interactions between the filovirus VP40 PPxY motifs and two unexpected novel interacting proteins, YAP (Yes-Associated Protein) and MAGI3 (Membrane Associated Guanylate Kinase 3; MAGUK). YAP is the main transcriptional activator of the Hippo signaling pathway, which regulates cell proliferation and apoptosis, and also antagonizes the host antiviral innate immune response by modulating the activity of a key transcription factor, IRF3. MAGI3 is a member of the MAGUK superfamily of proteins that functions as an adaptor/scaffolding protein at cell-cell junctions (tight junctions) to maintain junction integrity and barrier function. The interaction of both YAP and MAGI3 to the PPxY motifs of filovirus VP40 is particularly intriguing, as both of these proteins are also regulated by the host protein Angiomotin (Amot) following binding of their WW-domains to its N-terminal PPxY motifs. Amot functions as a direct negative regulator of YAP activity by binding and sequestering YAP at tight junctions. Amot also regulates tight junction assembly and integrity by interacting with the MAGI family of scaffolding proteins. Thus, Amot appears to be a ?master regulator? of Hippo signaling and tight junction formation. We hypothesize that VP40 mimics and competes with Amot for PPxY-mediated binding to YAP and MAGI3. We further hypothesize that: 1) VP40 binding to YAP prevents Amot from negatively regulating YAP activity, allowing for stabilization of YAP and its activity as an antagonist of the host innate antiviral response (Aim 1), and 2) VP40 binding to MAGI3 displaces Amot at tight junctions, leading to the dissolution of tight junction integrity and subsequent enhancement of permeability across barrier monolayers (Aim 2). Our results will provide innovative insights into the potential role of VP40 as a regulator of Hippo signaling, innate immune responses, and vascular leakage/permeability at tight junctions and immune privileged sites.